Human cytomegalovirus (CMV) is the major infectious cause of birth defects in the United States. Each year transmission of CMV from mother-to-fetus during pregnancy results in the birth of ~40,000 CMV-infected babies. While most suffer no overt ill effects from infection, ~8000 exhibit severe complications at birth and ~400 die within the first week of life. Of those that appear normal at birth another ~8000 may develop progressive and permanent hearing loss later in childhood. Both live attenuated and subunit CMV vaccines are under development. In both cases neutralizing antibodies to glycoprotein B (gB) have been emphasized as this protein is a key player in viral attachment and entry. However, past studies have measured the ability of antisera to block entry of CMV into fibroblast cells. Very recently it has been discovered that CMV can enter and infect endothelial, epithelial, and dendritic cells by an "endocytic" pathway that is quite distinct from the pathway used to infect fibroblasts. This pathway may be far more relevant in vivo than fibroblast entry. We hypothesize that viral entry into epithelial and endothelial cells may be blocked by antibodies specifically targeting viral proteins that mediate endocytic entry. Moreover, existing live attenuated vaccine strategies may lack efficacy because they fail to induce neutralizing antibodies specific for these endocytic-entry epitopes. Our objective is to promote development of an effective vaccine to prevent CMV-associated birth defects by (1) determining the extent to which neutralizing antibodies specifically targeting endocytic entry are induced by vaccination and natural infection;and (2) cloning the Townelong virus, which comprises a major component of the live attenuated Towne vaccine, defining its genetic composition, and characterizing the genetic and mechanistic basis of its cellular tropisms. The results of the proposed studies will guide the design of new vaccines or suggest modifications to existing vaccine strategies that have the potential to significantly improve vaccine efficacy. Project Narrative: Human cytomegalovirus (CMV) is the major infectious cause of birth defects in the United States. To promote development of an effective CMV vaccine, we propose to evaluate the importance of antibodies to recently identified proteins that are specifically involved in CMV entry into endothelial and epithelial cells and to determine the extent to which existing candidate vaccines are able to induce such antibodies and block epithelial/endothelial cell entry. The results of the proposed studies may reveal important shortcomings that could be addressed in the design of new vaccines or suggest modifications to existing vaccine strategies that will improve induction of antibodies targeting this entry pathway.